The regulation of glucose transport by insulin represents the rate limiting step in glucose utilization and storage, and is known to represent a primary lesion in patients with insulin resistance. The exocyst complex was found in our laboratory to play a role in the regulation of glucose transport, targeting Glut4 vesicles to sites of docking and fusion in fat cells. The overall goal of this proposal is to explore the molecular details of exocyst assembly and recognition, focusing on the key events that define its role in insulin- stimulated glucose transport. Our data suggest that the recognition of and release from the exocyst by the Glut4 vesicle-associated G protein RalA may be the last steps in an insulin-regulated G protein cascade controlling the itinerary of Glut4 trafficking. We propose that insulin stimulates the activity of Akt2, which phosphorylates the Ral GAP RGC, resulting in sequestration from its target G protein, RalA. We propose that RalA is thus activated, and then directs Glut4 vesicles along the final steps of the journey via actin cables to the exocyst, where it binds to the exocyst components Sec5 and Exo84. Once bound to RalA, Sec5 undergoes phosphorylation on Ser89 within the Ral binding domain, in the process releasing RalA from the exocyst and readying the vesicle for fusion. A round of dephosphorylation then recycles Sec5, allowing it to bind another vesicle-associated, active RalA. These findings suggest that the binding of the vesicle to the exocyst and its subsequent release might be the last regulated steps in Glut4 trafficking. We propose to explore this hypothesis by focusing on three specific aims: 1) identification of the molecular mechanisms by which Akt inactivates the Ral GAP complex, with a focus on understanding the role of phosphorylation; 2) understanding what controls the phosphorylation of the exocyst components to produce release of the Glut4 vesicle, concentrating on the exocyst protein Exo84 and the kinase TBK1; and 3) determining whether the Ral GAP complex plays an important in vivo role in glucose homeostasis by evaluating RGC knockout mice. We anticipate that these data will present a clearer picture of the role of the exocyst in the regulation of Glut4 trafficking, docking and fusion. PUBLIC HEALTH RELEVANCE: Glucose transport is the rate-limiting step in hormonally-regulated energy storage in muscle and fat cells, and is mediated by the glucose transporter Glut4. We will evaluate the role of the exocyst complex in this process, an evolutionarily conserved, eight protein targeting complex that ensures the localization of Glut4 in the cell.